RU2429292C1 - RECOMBINANT PLASMID pAg85A-CBD, Escherichia coli [pREP4, pAg85A-CBD] STRAIN, CHIMERIC PROTEIN Ag85A-CBD AND THEIR APPLICATION - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: synthetic gene Ag85A Mycobacterium tuberculosis optimised for heterologous expression in nonpathogenic laboratory strains. The recombinant plasmid pAg85A-CBD consisting of an artificial bacterial operon of chimeric protein containing a promotor area of an early promotor of bacteriophage T5, chimeric protein gene and transcription terminator, a bacterial operon of beta-lactamase and a bacterial ColEl-type replication initiation site is produced. The invention also involves the Escherichia coli strain producing chimeric protein Ag85A-CBD, and also a method of immobilisation, concentration and cellulose purification of produced protein. Besides, the invention refers to the same recombinant protein Ag85A-CBD and an immunogenic composition containing it and used for tuberculous infection immunity induction.

EFFECT: invention allows preparing the producer strain providing a high production level of resistant immunogenic proteins which can be one-stage produced, immobilised and purified, producing the effective immunogenic compositions for tuberculosis.

7 cl, 2 dwg, 5 ex

Description

The invention relates to genetic engineering, biochemistry, biotechnology and immunology.

Currently, the only vaccine used in the world against tuberculosis is BCG (BCG, Bacillus Calmette-Guerin, 1921), which is a live, attenuated strain of Mycobacterium bovis. It is safe, inexpensive and protects children quite effectively both from infection with tuberculosis and from miliary tuberculosis and tuberculous meningitis. However, BCG does not protect adults from pulmonary tuberculosis, namely this form of the disease leads to the spread of infection and a serious epidemic. Hence the high relevance of the development of new vaccines against tuberculosis.

There are several directions for the development of new TB vaccines, alternative to BCG: subunit vaccines, DNA vaccines and live vaccines with improved properties. Subunit vaccines are most fully characterized in experimental animal models, including preparations consisting of several secreted proteins / peptides of Mycobacterium tuberculosis (Doherty T.M., Olsen AW, Weischenfeldt 1 J. et al. Comparative Analysis of Different Vaccine Constructs Expressing Defined Antigens from Mycobacterium tuberculosis. The Journal of Infectious Diseases, 2004, Vol. 190, pp. 2146-2153). Many M. tuberculosis antigens have been identified, potentially important as components of new vaccines. These include, in particular, immunodominant secreted antigens present in M. tuberculosis culture filtrate. Among the secreted M. tuberculosis antigens, ESAT-6 (Early Secret Antigen Target-6), CFP-10 (culture-filtrate protein-10) and complex 85 proteins (Ag85A, B, C) are best studied.

The closely related proteins Ag85A, B, C are encoded by three separate genes present in many strains of mycobacteria, including the BCG vaccine strain (Wiker HG, Harboe M. The Antigen 85 Complex: a Major Secretion Product of Mycobacterium tuberculosis. Microbiol. Reviews.,. 1992, Vol. 56, No.4, pp. 648-661). Antigens 85 are also involved in the formation of a T-cell response in individuals infected with M. tuberculosis (Launois P, DeLeys R, Niang MN, et al. T cell epitope mapping of the major secreted mycobacterial antigen Ag85A in tuberculosis and leprosy. Infect. Immun. 1994, Vol.62, pp.3679-3687).

The objective of the invention is to develop vaccines based on individual protein components with a high level of expression and stability of these proteins in the bacterial system, with a high degree of purification and stability of the components.

To solve this problem, we use the design of chimeric proteins by combining two genes in one translation frame — a gene for a vaccinating protein and a gene for a carrier protein, which leads to the synthesis of a chimeric protein in the bacterial system. Recombinant proteins were created consisting of two components: M. tuberculosis antigen and the cellulose-binding domain of CelD from the thermophilic microorganism Anaerocellum thermophilum (CBD). Recombinant proteins containing CBD can be immobilized in a single step on a cellulose carrier. The result is their simultaneous purification, concentration and stabilization, as well as protection against proteases of producer bacteria. In this case, the initial antigenic properties of the functionally active protein domain are not violated.

In addition, a synthetic Ag85A gene was obtained. To ensure a high level of expression of this gene in a heterologous system, codons were optimized. As a result, the synthetic Ag85A gene was planned, which differed from the native mycobacterial gene in nucleotide composition with the preserved amino acid sequence of the Ag85A protein encoded by it. This technique made it possible to obtain a highly efficient producer strain synthesizing up to about 15-20% of the recombinant Ag85A antigen from the total protein of the cell.

Using these approaches, a two-component recombinant protein was obtained. The sequence of the M. tuberculosis gene Ag85A encodes a full-sized protein that forms the first protein domain that determines the functional immunological properties of the complex molecule. The following is the connecting (spacer) amino acid sequence of several alternating glycine and serine residues (Gly-Ser). The second protein domain is encoded by the A.thermophilum CelD endoglucanase gene sequence; it determines the ability of the product to interact with the cellulosic sorbent. Protein products are produced in non-pathogenic laboratory E. coli strains. Antigens immobilized on cellulose are a suspension of a sorbent with proteins adsorbed on it. The cellulose carrier acts as an adjuvant in immunization, which provides antigen enlargement and polymerization, without which a weaker immune response to mono-protein antigens develops. Free antigens are solutions with a certain ionic strength, free from impurities of lipopolysaccharides and DNA of the producer strain.

Purification of end products from highly toxic lipopolysaccharides, ballast proteins, DNA and RNA of producer strains is an important problem.

Thus, the claimed group of inventions allows to obtain the optimal synthetic gene Ag85A for heterologous expression, to create an E. coli strain that provides a high level of production of a recombinant protein containing M. tuberculosis Ag85A protein; to obtain a simple and effective scheme for purification of a recombinant protein that specifically and effectively induces an immune response to mycobacterial antigens, including the synthesis of IFN-gamma, necessary for anti-tuberculosis protection; to create immunogenic compositions based on a recombinant protein on a cellulose carrier (adjuvant).

This result is achieved through the synthesis of the recombinant protein Ag85A-CBD in the cells of the recombinant strain E. coli M15 [pREP4, pAg85A-CBD], carrying the recombinant plasmid pAg85A-CBD. Also, by creating a complex preparation Ag85A-CBD-cellulose, in which the recombinant protein Ag85A-CBD (concentration 5.21 mg / ml) is immobilized on cellulose (2.8% by weight in the preparation) and resuspended in 1 × PBS pH 7 buffer , 2-7.4.

The essence of the invention lies in the fact that the plasmid contains the following structural elements essential for its functioning:

a) a synthetic gene encoding the sequence of M. tuberculosis Ag85A protein (912 bp);

b) an artificial bacterial operon of a chimeric protein, consisting of:

- the promoter region of the early promoter of the T5 bacteriophage (7-87 bp), which provides efficient transcription of mRNA;

- a recombinant gene encoding a chimeric protein "M. tuberculosis Ag85A protein sequence - spacer - A. thermophilum cellulose-binding domain" - Ag85A-CBD (117-1628 bp);

- the untranslated region of the termination of transcription of the bacterial operon, which ensures the effective termination of transcription of pAg85A-CBD (1659-1665 bp);

c) the bacterial operon bla encoding beta-lactamase protein, a selective marker for the transformation of the E. coli strain pAg85A-CBD (3826-4686 bp) of the complementary chain;

d) a bacterial site of ColE1 type replication initiation, which provides plasmid replication in the E. coli strain: pAg85A-CBD (3063-3074 bp).

Thus, a recombinant plasmid pAg85A-CBD (4891 bp) was created that encodes a bifunctional recombinant protein Ag85A-CBD, which has the ability to spontaneously bind to a cellulose-containing sorbent. The plasmid contains the following structural elements: an artificial bacterial operon of the chimeric protein, including the promoter region of the early promoter of the T5 bacteriophage (80 bp), the gene of the chimeric protein Ag85A-CBD (1511 bp) and the transcription terminator (94 bp) ; beta-lactamase bacterial operon (860 bp) and ColE1 type bacterial replication initiation site (11 bp). The recombinant protein producing strain Ag85A-CBD is obtained by transforming E. coli cells of strain M15 [pREP4] with plasmid pAg85A-CBD. The strain provides the production of recombinant protein Ag85A-CBD after the induction procedure (isopropyl-β-D-thio-galactopyranoside) (IPTG) up to 12-15% of the total protein of the cell.

Strain E. coli M15 [pREP4], carrying the plasmid pAg85A-CBD, the producer of the bifunctional recombinant protein Ag85A-CBD is characterized by the following features.

Cultural and morphological characters. Cells are straight, rod-shaped, motionless, gram-negative. When sieving on a plate with 2.0% agarized LB medium, growth is in the form of separate colonies, sometimes in the R-form with uneven edges. It grows well on solid and liquid nutrient media (LB-broth, LB-agar, MPA, MPB).

Physiological and biochemical characteristics. Cells grow at temperatures from + 4 ° C to + 42 ° C, with an optimum pH of 6.8-7.5. The strain decomposes glucose, mannitol with the formation of acid, does not decompose sucrose, arabinose, galactose, ferments maltose, xylose, sorbitol, rhamnose. Significant when using this strain is its sensitivity to nalidixic acid (25 mg / ml), streptomycin (20 mg / ml) and rifampicin (25 mg / ml). It is resistant to ampicillin (up to 300 μg / ml), due to the presence of plasmid pAg85A-CBD, and to kanamycin (up to 50 μg / ml), due to the presence of plasmid pREP4.

To create a complex preparation of recombinant protein and amorphous cellulose, the technical result is achieved by creating a two-component recombinant protein Ag85A-CBD, consisting of M. tuberculosis Ag85A protein and CelD A.thermophilum endogluconase gene. And also by obtaining a complex preparation (immunological composition) in which a recombinant protein (Ag-CBD) is immobilized on amorphous cellulose (Ag-CBD-cellulose).

The recombinant protein Ag85A-CBD contains a domain that determines its ability to bind to cellulose, which allows concentration, purification and immobilization of the protein product on cellulose in one step. Cellulose immobilization is ensured by the presence in the recombinant protein of the cellulose-binding domain of the CelD endogluconase from the thermophilic microorganism A. thermophilum, which has a high affinity for crystalline and amorphous celluloses and provides irreversible binding to the carrier in a wide range of pH 4-11, temperatures: from 0 to + 75 ° C and NaCl concentrations: from 0 to 5 M.

Since cellulose binding proteins are absent in E. coli, the recombinant protein Ag85A-CBD synthesized in E. coli cells transformed with pAg85A-CBD plasmid is the only protein of the producer strain that binds strongly to cellulose. This provides the possibility of a single-stage preparation of a highly purified protein preparation immobilized on a sorbent.

The immunogenicity of the complex preparation Ag85A-CBD-cellulose in mice was studied using two methods. First, mice were immunized in the paw pads of the hind legs and after 18 days the proliferative response of T cells was compared with the response of mice that were only immunized with CBD cellulose. An expressed specific immune response to the antigen was revealed, with experience proliferation indices: control = 5-7. In another design, mice were immunized under the skin of the back 2 times with a 2-week interval immunogenic and control drug. After 5 weeks, the proliferative response of T cells and the number of T cells producing IFN-gamma were investigated. A 2-3-fold increase in IFN-gamma-positive T cells and a 4-fold increase in proliferation in the presence of antigen in cell culture were detected compared with control animals.

To test the protective activity of the studied drugs, they are immune to the Ag85A antigen (immunization with Ag85A-CBD-cellulose), as well as control animals (immunization with CBD-cellulose and saline) were infected with an intravenously virulent M. tuberculosis H37Rv strain at a dose of 5 × 10 ⁶ CFU / mouse. 3 weeks after infection, 4 mice from each group were scored and organ homogenates were seeded on Petri dishes with Dubo agar. In the lungs and spleens of mice immune to Ag85A, a 5-fold decrease in bacterial load was observed compared to control animals.

The technical result achieved by the implementation of the invention is to obtain a producer strain that provides a high level of production of a stable immunogenic protein that can be obtained, immobilized and purified in one stage. An effective immunogenic composition was obtained against tuberculosis.

The invention is illustrated by the following examples below. Genetic engineering and microbiological manipulations, amplification and DNA sequencing were performed according to standard methods (T. Parish, NGStoker. Mycobacterium tuberculosis protocols., Humana Press Inc., 2001, Methods in molecular Medicine, 54; Maniatis T., Fritsch E., Sambrook J. Molecular Cloning, M .: Mir, 1984; DNA Cloning, Methods, Edited by D. Glover, Translated from English, M .: Mir, 1988; Saiki RK, Gelfand DH, Stoffel S. et al. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science, 1988, Vol. 239, No. 4839, pp. 487-491; Sanger F., Nicklen S., Coulson AR DNAsequencing with chain-terminating inhibitors (DNA polymerase (nucleotide sequences / bacteriophage 4X174). Proc. Nat. Acad. Sci. USA, 1977, Vol. 74, No.12, pp. 5463-5467).

Example 1. Obtaining a plasmid.

a) Obtaining a gene fragment of the protein M. tuberculosis.

The synthetic gene of the Ag85A protein was designed based on the protein sequence so that the nucleotide composition of the codons was optimized for heterologous expression in the non-pathogenic laboratory E. coli strain, and the amino acid composition of the synthesized recombinant protein was not changed. To clone a DNA fragment, the gene sequence was flanked by restriction endonucleases BamHI, BspMII (Kpn2I) (data not shown).

A fragment of the Ag85A protein gene was synthesized by the solid-phase amidophosphite method (ZAO Evrogen, RF) using an Applied Biosystems ABI 3900 synthesizer, followed by cloning of the duplex DNA into a pAL-TA vector. The primary structure of the resulting construct was confirmed by sequencing. For subsequent cloning of the ag85A gene fragment, the restriction endonucleases BamHI, BspMII (Kpn2I), BglII, PvuI, as well as translation initiation and termination codons, were introduced into the expression vector in its sequence.

b) Obtaining a plasmid containing the gene sequence of the mycobacterial protein, the Gly-Ser spacer and the cellulose-binding domain of CBD.

To obtain a construct encoding the gene sequence of the M. tuberculosis protein Ag85A protein, Gly-Ser spacer, and the cellulose-binding domain of CBD, plasmid pALTA-Ag85A was hydrolyzed with restriction endonucleases BglII and PvuI at 37 ° C in a buffer containing 50 mM Tris-HCl (pH 7.5), 10 mM magnesium chloride, 100 mM sodium chloride and 0.1 mg / ml BSA, for 1 hour. Plasmid pspCBD (ptt10) encoding the sequence of Gly-Ser and the cellulose-binding domain was digested with restriction endonucleases BamHI and BglI at 37 ° C in a buffer containing 66 mM Tris-acetate (pH 7.9 at 37 ° C), 20 mM acetate magnesium, 132 mM potassium acetate and 0.2 mg / ml BSA for 1 hour. Restriction products were separated on a 1.2% agarose gel. The restriction fragments of plasmid DNA pALTA-Ag85A (1630 bp) and pspCBD (3261 bp) isolated from the agarose gel were ligated using T4 bacteriophage DNA ligase. Using electroporation, the obtained ligase mixture was used to transform competent E. coli M15 cells [pREP4]. After transformation, clones were selected on LB agar medium containing antibiotics ampicillin and kanamycin. Plasmid DNA was isolated by alkaline lysis, analyzed by restriction mapping with restriction endonucleases BamHI, BglI, BgLII, NcoI, PvuI. Clones of plasmid DNA pAg85A-CBD (4891 bp) containing the sequence of the tuberculosis protein, Gly-Ser spacer, and CBD cellulose-binding domain were selected. The primary structure of the resulting construct was confirmed by sequencing. Figure 1 shows a diagram of the plasmid pAg85A-CBD.

Example 2. Construction of a strain of E. coli - producer of the recombinant antigen M. tuberculosis, connected to the cellulose-binding domain.

To obtain the E. coli strain, a producer of the recombinant protein Ag85A-CBD, cells of the E. coli strain M15 [pREP4] were transformed with the plasmid pAg85A-CBD. Transformed cells were grown in 3.5 ml of LB medium with ampicillin and kanamycin at 37 ° C to an optical density corresponding to 1 unit. absorption at a wavelength of 550 nm (about 2.5 hours). 3 μl of a 0.1 M solution of isopropyl-beta-D-thiogalactopyranoside (IPTG, IPTG) was added to the culture and grown for 3 h. Electrophoresis was used to control the production of the recombinant protein in E. coli M15 strain [pREP4, pAg85A-CBD] according to Laemmli in the presence of sodium dodecyl sulfate (SDS). Protein separation was carried out on a 12% polyacrylamide gel (PAGE) in a standard buffer system (electrode buffer: 25 mM Tris-HCl, 192 mM glycine, 0.1% sodium dodecyl sulfate, pH 8.3; gel buffer: 375 mM Tris-HCl pH 8.8). At the end of electrophoresis, the gels were stained with 0.15% Coomassie G250 solution in 25% isopropanol and 10% acetic acid and washed in 10% acetic acid.

When comparing the spectrum of proteins in strains of E. coli M15 [pREP4], E. coli M15 [pREP4, pAg85A-CBD], the appearance of an additional protein band was detected, the molecular weight of which was 54.1 kDa, which corresponded to the calculated value for the protein Ag85A-CBD. The level of protein synthesis in E. coli was determined by comparing the staining intensity of the recombinant protein band with the band of the corresponding Unstained Protein Molecular Weight Marker standard protein (Fermentas, Lithuania).

To test the solubility of the synthesized protein, the biomass of the grown E. coli M15 strain [pREP4, pAg85A-CBD] was destroyed in 1% TES buffer (25 mM Tris-HCl, 5 mM EDTA, 50 mM NaCl, 1% Triton X-100, 5 mg / ml lysozyme) in the ratio of biomass-buffer 1: 2 and homogenized to a homogeneous mass. After centrifugation, the samples were divided into 2 fractions: soluble cellular proteins (supernatant) and insoluble (precipitate). Both fractions were degraded in lysis buffer (0.05 M Tris-HCl, pH 6.8, 0.5% Triton X-100, 0.5 M NaCl, 0.25 M MgCl ₂ , 5 mg / ml lysozyme, 10 mg / ml PMSF, 20 mg / ml DNase) in a 1: 1 ratio; homogenized to a homogeneous mass; warmed at + 95 ° C for 15 minutes The results were monitored by Lammley electrophoresis. It was shown that the recombinant protein Ag85A-CBD is synthesized in E. coli cells both in the soluble fraction and in the form of Taurus inclusions.

Example 3. Obtaining recombinant protein Ag85A-CBD in a free state and immobilized on cellulose.

To obtain a recombinant protein, a culture of E. coli strain M15 [pREP4, pAg85A-CBD] was grown in 1000 ml of LB medium with ampicillin and kanamycin at 37 ° C to an optical density corresponding to 1 unit. absorption at a wavelength of 550 nm. 100 μl of a 0.1 M IPTG solution was added to the medium and grown for 3 hours. Cells were pelleted by centrifugation at 5500 rpm for 15 minutes.

The pellet was resuspended in lysis buffer containing 50 mM Tris-HCl (pH 8.0), 0.25 mM NaCl, 5 mM MgCl ₂ , 0.15 mM PMSF, 0.1% Triton-X100, 0.5 mg / ml lysozyme, 20 mg / ml DNase; based on 1 g of biomass per 4 ml of buffer. Additionally, the suspension was treated with ultrasound 3 times for 20 seconds. After centrifugation at 16,000 rpm for 30 min, the fraction of insoluble cellular proteins remained in the sediment, soluble in the supernatant.

The precipitate (insoluble fraction) was suspended in lysis buffer (50 mM Tris-HCl (pH 8.0), 10 mM EDTA, 0.002 volume of β-mercaptanol) and an equal (by weight) volume of urea was added. After mixing, it was kept in a water bath at + 37 ° C until the urea was completely dissolved. It was centrifuged at 12,000 rpm for 30 minutes and the supernatant was taken. To purify recombinant proteins, the supernatant was transferred onto a microcrystalline cellulose column (Perloza MT-200, Iontosorb, Czech Republic). Protein elution from cellulose was carried out with a solution of 8–2 M urea and 50 mM Tris-HCl (pH 8.0). To remove urea, the samples were dialyzed against PBS buffer at 25 ° C. overnight.

The soluble protein fraction was used for immobilization on injection (amorphous) cellulose. 1/3 of the volume of a 30% suspension of cellulose and a buffer containing 0.5% Triton X-100, 0.5 M NaCl were added to the supernatant; incubated at 25 ° C for 1 h. Centrifuged for 10 min at 8000 rpm, the pellet was resuspended in the same buffer; cellulose washing was repeated 2 times. The pellet was resuspended in PBS buffer. In a state immobilized on a sorbent, proteins are a suspension.

The concentration of proteins in free and immobilized forms was determined by Bradford at a wavelength of 595 nm. To determine the% cellulose content in the samples, where the proteins were immobilized on a sorbent, the samples were lyophilized for two days, followed by recounting of the obtained masses. Thus, the following complex preparations (immunogenic compositions) were obtained (Ag-CBD, Ag-CBD-cellulose), the purity of which was not less than 95%:

No. p / p Sample Name The concentration of protein in the sample, mg / ml % cellulose in the sample one Ag85A-CBD 5.18 no 2 CBD 1.36 no 3 Ag85A-CBD Cellulose 5.21 2.8 5 CBD pulp 5,6 2,8 6 cellulose no 2,8

Example 4. A method for checking the immunogenicity of complex preparations of Ag-CBD-cellulose in mice.

Female mice of an inbred line of I / St mice genetically sensitive to tuberculosis (Nikonenko BV, Averbakh MM, Lavebratt C, Schurr E, Apt AS. Comparative analysis of mycobacterial infections in susceptible I / St and resistant A / Sn inbred mice. Tubercle Lung Dis , 2000, 80: 15-25), the following preparations were injected subcutaneously at a two-week interval: 1) Ag85A-CBD-cellulose (10 μg per mouse according to Ag85A content); 2) CBD cellulose (10 μg per mouse according to CBD content) - negative control; 3) a single vaccine BCG (10 ⁶ CFU per mouse) - a positive control. After 5 weeks, the content in the spleens of T-lymphocytes producing IFN-gamma in the presence of the corresponding antigens in the culture fluid was evaluated by ELISPOT method, with counting of cells reacting with anti-IFN-gamma antibodies, 60 hours after the start of cultivation. The results are shown in the table:

Vaccination The number of IFN-gamma producers per 1 million spleen cells No antigen in culture 10 μg / ml antigen in culture * Ag85A-CBD Cellulose 10 ± 3 45 ± 14 BCG 31 ± 3 70 ± 7 CBD pulp 4 ± 2 17 ± 3 * In the control groups (BCG and CBD cellulose vaccination), the ultrasonic disintegrate of mycobacteria was used as antigen, i.e. a mixture of many antigens, since BCG stimulation is polyclonal.

The results show that vaccination with monoantigens bound to cellulose through the CBD domain leads to specific activation of IFN-gamma producers. Vaccination with the same antigens, in the same quantities, but without adsorption on cellulose, does not cause an immune response (data not shown).

Example 5. A method for checking the protective effect against tuberculosis of complex preparations of Ag-CBD-cellulose in mice.

C57BL / 6 inbred mice were vaccinated with Ag85A-CBD-cellulose preparations (10 μg per mouse by Ag85A content); 2) ESAT-6-CBO-cellulose (10 μg per mouse according to the content of ESAT-6); 3) CBD cellulose (10 μg per mouse according to CBD content - negative control 1); 4) with physiological saline (negative control 2) twice, subcutaneously, with an interval of 2 weeks. 5 weeks after the second vaccination, the mice were infected intravenously at a dose of 5 × 10 ⁶ CFU of the virulent M. tuberculosis H37Rv strain. 4 weeks after infection, 4 mice were scored from each group and the multiplication of mycobacteria in the lungs and spleen was assessed by seeding organ homogenates on Dubo agar followed by colony counting. The remaining mice (8 in each group) were observed to determine survival curves. The results are shown in figure 2. It was found that vaccination with specific antigens on a cellulosic sorbent significantly reduces the number of mycobacteria in organs (Fig. 2A, P <0.01, unpaired Student's test) and prolongs the life of infected animals (Fig. 2B, P <0.01, Gohanan test for curves survival). Evaluation of the parameters of the immune response in mice:

- Proliferative activity of CD4 ⁺ T-lymphocytes in in vitro tests;

- The increase as a result of immunization, the number of cells of the immune system producing IFN-gamma;

- Reducing the number of virulent mycobacteria in the lungs and spleen of infected mice;

- Increased survival of mice after infection.

Claims (7)

1. Synthetic gene Ag85A M. tuberculosis (912 bp), encoding the protein Ag85A M. tuberculosis, with nucleotide sequence No. 1, with codons optimized for heterologous expression in E. coli strain. 2. Recombinant plasmid pAg85A-CBD with nucleotide sequence No. 2, which provides expression of the recombinant protein Ag85A-CBD, consisting of the recombinant protein Ag85A M. tuberculosis, Gly-Ser spacer, cellulose-binding domain of the endoglucanase CelD gene from A.thermophilum, size 48, size 48. ., consisting of the following structural elements:
- an artificial bacterial operon of the recombinant protein Ag85A-CBD, including: the promoter region of the early promoter of the bacteriophage T5 (7-87 bp), the synthetic gene of the recombinant protein Ag85A-CBD (117-1628 bp), the untranslated region of transcription termination ( 1665-1759 bp);
- the bacterial operon beta-lactamase, which provides resistance to ampicillin (3826-4686 bp);
- a bacterial site of replication initiation type ColE1 (3063-3074 bp), which ensures replication of the plasmid in E. coli strains. 3. Recombinant E. coli strain M15 [pREP4, pAg85A-CBD], obtained by transformation with the plasmid according to claim 1, producer of the protein Ag85A-CBD, deposited in VKPM under the number VKPM 10534. 4. The method of obtaining, immobilization, concentration and purification of recombinant protein Ag85A-CBD on cellulose, including:
- growing cells of E. coli M15 strains [pREP4, pAg85A-CBD] according to claim 2;
- immobilization of Ag85A-CBD protein as part of cell extracts of E. coli strain M15 [pREP4, pAg85A-CBD] on a cellulose sorbent due to affinity interaction during the incubation procedure followed by washing from unbound bacterial proteins, and the target product is concentrated and purified;
- selection of the target product. 5. Recombinant protein Ag85A-CBD with a molecular weight of 54.1 kDa (pI 5.88), including a full-sized Ag85A protein with sequence No. 3, Gly-Ser spacer with sequence No. 4, cellulose-binding domain of the A. thermophilum endoglucanase gene CelD with sequence No. 5. 6. Immunogenic composition containing the recombinant protein according to claim 5 of Ag85A-CBD, immobilized on cellulose in an effective amount, specifically activating T lymphocytes that synthesize IFN-gamma upon stimulation with mycobacterial antigens. 7. A method of checking the protective properties of the immunogenic composition according to claim 5 in relation to tuberculosis infection in a model of tuberculosis in mice, including:
a) vaccination of C57BL / 6 inbred mice twice, subcutaneously, with an interval of 2 weeks with preparations: immunogenic composition according to claim 5 at the rate of 10 μg per mouse according to the content of the active substance; 10 μg CBD cellulose per mouse based on CBD content - negative control 1; saline - negative control 2;
b) infection 5 weeks after the second vaccination of mice intravenously at a dose of 5 × 10 ⁶ CFU with a virulent strain of M. tuberculosis H37Rv;
c) killing 4 mice from each group 4 weeks after infection from each group and assessing the multiplication of mycobacteria in the lungs and spleen by plating organ homogenates on Dubo agar followed by colony counting;
d) conducting observations of the remaining mice (8 in each group) to construct survival curves;
e) in this case, the protective properties of the immunogenic composition according to claim 5 are judged by the results of stages c) and d), comparing them between experience and control within each group, while reducing the number of mycobacteria in organs and prolonging the life of infected animals indicates the presence of the composition has protective properties.

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